Adult tissues that comprise the organs of the body undergo a constant turnover of their cells. Typically, senescent terminally differentiated cells are removed by cell shedding, necrosis or by programmed cell death (apoptosis), and are replaced by cells derived from tissue specific stem- or progenitor cells. Similarly, tumors have been shown to be composed of terminally differentiated as well as a small fraction of stem-like cells. It is this latter subpopulation that is believed to drive tumor growth, play a role in metastasis, and to cause resistance to therapy through its regenerative capacity. However, although well established as a concept, tumor stem cells are notoriously difficult to identify, isolate and characterize. To date, no unique marker for cancer stem cells has been described. Furthermore, it is not yet known if tumor stem cells are tissue stem cells that have acquired malignant potential, or if they are differentiated malignant cells that have been genetically reprogrammed to express the genes characteristic of the tissue stem cell phenotype. Recently, it has been shown that (1) increased rigidity of the extracellular matrix causes phenotypically malignant growth of epithelial cells, (2) stem cell differentiation is strongly influenced by the elasticity of the extracellular matrix, (3) increased tissue density increases breast cancer risk, and (4) wounding increases matrix elasticity and tumor growth. For this project, we propose that the elasticity of the extracellular matrix influences tumor development and growth by providing a pathophysiological mechanism for the development of stem cell like malignant epithelial cells (tumor stem cells). We are currently focusing on the stem cell subpopulation of normal breast ductal epithelial cells and breast cancer stem cells as a model to elucidate this mechanism. During fiscal year 2007 we evaluated aldehyde dehydrogenase activity in combination with cell surface markers such as CD15 and CD133 as a tool to segregate subpopulations in tumor cell lines (using the NCI 60 cell line panel). We developed and established isolation and culture methods for ductal epithelial and stromal cells of the normal human breast in these short term cultures using FACS analysis and immunofluorescence. We established protocols for generating acrylamide and silicone matrices of defined elasticity that can be functionalized with matrix components such as fibronectin. Preliminary experiments using cell lines indicate that with increasing matrix density breast epithelial cells indeed show alterations of protein expression that are typically found in tumor cells.